Gene Silencing by Nuclear Orphan Receptors

1 Gene Silencing by Nuclear Orphan Receptors Ying Zhang and Maria L. Dufau Section on Molecular Endocrinology, Endocrinology, and Reproduction Research Branch, National Institutes of Health Bethesda, Maryland 20892

I. Introduction II. COUP-TFI, II (EAR3, ARP-1), and EAR2: Inhibitors of Diverse Genes by Multiple Mechanisms III. DAX-1: Silencing in the Control of Steroidogenic and Sex-Determining Target Genes IV. GCNF: Negative Control during Gametogenesis and Embryonic Development V. SHP: Generic Heterodimeric Partner-Inhibiting Multiple Nuclear Receptor Pathways VI. TR4 and TR2: Testicular Receptor with Homologous but Not Redundant Functions VII. Conclusions References

Nuclear orphan receptors represent a large and diverse subgroup in the nuclear receptor superfamily. Although putative ligands for these orphan members remain to be identiWed, some of these receptors possess intrinsic activating, inhibitory, or dual regulatory functions in development, diVerentiation, homeostasis, and reproduction. In particular, gene-silencing events elicited by chicken ovalbumin upstream Vitamins and Hormones Volume 68

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Copyright 2004, Elsevier Inc. All rights reserved. 0083-6729/04 $35.00

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promoter-transcription factors (COUP-TFs); dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1); germ cell nuclear factor (GCNF); short heterodimer partner (SHP); and testicular receptors 2 and 4 (TR2 and TR4) are among the best characterized. These orphan receptors are critical in controlling basal activities or hormonal responsiveness of numerous target genes. They employ multiple and distinct mechanisms to mediate target gene repression. Complex cross-talk exists between these orphan receptors at their cognate DNA binding elements and an array of steroid/nonsteroid hormone receptors, other transcriptional activators, coactivators and corepressors, histone modiWcation enzyme complexes, and components of basal transcriptional components. Therefore, perturbation induced by these orphan receptors at multiple levels, including DNA binding activities, receptor homo- or heterodimerization, recruitment of cofactor proteins, communication with general transcriptional machinery, and changes at histone acetylation status and chromatin structures, may contribute to silencing of target gene expression in a speciWc promoter or cell-type context. Moreover, the Wndings derived from gene-targeting studies have demonstrated the signiWcance of these orphan receptors’ function in physiologic settings. Thus, COUP-TFs, DAX-1, GCNF, SHP, and TR2 and 4 are known to be required for multiple physiologic and biologic functions, including neurogenesis and development of the heart and vascular system steroidogenesis and sex determination, gametogenesis and embryonic development, and cholesterol/lipid homeostasis. ß 2004 Elsevier Inc.

I. INTRODUCTION The largest superfamily of eukaryotic transcription factors comprises nuclear receptors and contains more than 150 proteins, including steroid/ nonsteroid nuclear hormone receptors and orphan receptors (Mangelsdorf et al., 1995; Perlmann and Evans, 1997). The nuclear hormone receptors are ligand-inducible transcripton factors that have critical roles in the control of development, diVerentiation, homeostasis, and reproduction. These diverse actions are initiated by their speciWc binding to small lipophilic molecules such as steroid and thyroid hormones, retinoids, and vitamin D3 (Harvey and Williams, 2002; Haussler et al., 1997; Ruberte, 1994). The majority of nuclear receptors are orphan receptors for which no ligand was initially identiWed (Giguere, 1999). During recent years, the identiWcation of novel ligands for a number of these receptors, which include the retinoid X receptor (RXR) (Mangelsdorf et al., 1992), peroxisome proliferatoractivated receptor (PPAR) (Devchand et al., 1999), farnesoid X receptor (FXR), (Niesor et al., 2001; Tu et al., 2000), liver X receptor (LXR) (Peet et al., 1998), and pregnane X receptor (PXR) (Honkakoski et al., 2003), has

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led to the characterization of their functional roles and an understanding of their signiWcance in metabolic regulation (Fitzgerald et al., 2002). At this stage, it is not known whether the numerous unliganded orphan receptors exert their diverse actions in a ligand-independent manner or are regulated by speciWc ligands that are yet to be identiWed. Since nuclear orphan receptors occur in nearly all species examined and outnumber the steroid/ nonsteroid hormone receptors, they probably represent an ancient and complex class of regulatory proteins that participate in multiple physiologic functions. Many nuclear orphan receptors exert constitutively active regulation on numerous target genes through their intrinsic activation/ repression activities or through multiple modes of cross-talk with nuclear hormone receptors. This chapter focuses on the silencing mechanisms of nuclear orphan receptor-mediated regulation of gene expression. To facilitate the understanding of down-regulation in the context of a nuclear receptor superfamily, a brief account of the structural organization of nuclear receptors in relationship with their function is presented. Nuclear receptors are modular proteins and consist of Wve characteristic domains (Fig. 1A). These include an N-terminal region (A/B), a DNA binding domain (C), a hinge region (domain D), and C-terminal ligand binding domains (E and F) (Evans, 1988). The A/B domain is highly variable among the members of the nuclear receptor family. It possesses a ligand-independent transcriptional activation function, which is referred to as AF-1. The AF-1 mediated gene activation often displays promoter-dependent and cell-type-dependent speciWcities, indicating that this domain may be responsible for interactions with cell-speciWc coregulatory proteins (Bastien et al., 2000; Rochette-Egly et al., 1997). Domain C, the DNA binding domain (DBD), is highly conserved and is a signature motif of the nuclear receptor superfamily. The DBD targets receptors to speciWc DNA sequences, known as hormone response elements (HREs), in the promoter region of target genes. Two zinc Wnger motifs in this domain serve as an interaction surface to contact a hexameric DNA core sequence containing nucleotides AGGTCA (Freedman and Towers, 1991; Umesono and Evans, 1989). The unique actions of nuclear receptors are highly speciWc and dependent on DNAreceptor interactions and on receptor-receptor dimerization. Accordingly, they usually bind as homodimers or heterodimers to two copies of the response elements that can be arranged as inverted (palindromic), everted, or direct repeats. Receptors for estrogen or glucocorticoid steroid hormones recognize palindromic HREs as homodimers (Evans, 1988). In contrast, most nonsteroid receptors heterodimerize with RXR at various direct-repeat motifs, and the spacing between the two repeats often dictates the binding speciWcities of diVerent heterodimers (Mangelsdorf and Evans, 1995). Such heterodimers have been shown to function as dynamic transcription factors in which the two partners influence each other’s ability to interact with ligands and cofactors. The DBDs also serve to stabilize receptor

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FIGURE 1. Schematic structure of a nuclear receptor and the ligand binding domain (LBD) in absence or presence of a ligand. (A) A typical nuclear receptor with its Wve characteristic domains. The N-terminal A/B domain is a variable among nuclear receptors family members and harbors a ligand-independent activation function (AF-1). The domain C represents the highly conserved DNA binding domain (DBD), which is the signature motif of the nuclear receptors. The hinge domain D as a nonconserved linker region connects the DBD to the C-terminal E and F domains where E is the LBD binding domain (LBD) responsible for multiple functions of the receptor. The ligand-inducible activation function (AF-2) is located at the C-terminal of the LBD. (B, Left) Crystal structure of LBD of the unligand human RXR
receptor. (B, Right) Crystal structure of LBD of the RARg receptor bound to all-trans retinoic acid. There are 12
-helices (numbered H1 to H12) that are indicated by cylinders, where the orientation and position of H12 (containing AF-2) undergoes signiWcant changes after ligand binding (Bourguet et al., 1995; Renaud et al., 1995). Reprinted with permission from Nature.

dimerization on the DNA template and have a fundamental role in DNA sequence recognition (Rastinejad et al., 1995). Moreover, several nuclear orphan receptors, including steroidogenic factor-1 (SF-1), nerve growth factor inducible-B (NGFI-B) nuclear receptor, and retinoid-related orphan

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receptor (ROR) bind to their DNA targets as monomers; the binding requires an A/T-rich sequence 50 adjacent to the core motif (Giguere et al., 1995; Meinke and Sigler, 1999; Wilson et al., 1993). Domain D, the hinge region that links the DBD and ligand binding domain (LBD), is not well conserved and varies signiWcantly in length. This domain has been proposed to participate in DNA rotation and corepressor interaction (McBroom et al., 1995). The C-terminal region of nuclear receptors is a multifunctional domain and mediates ligand binding, receptor dimerization, and transcriptional activation or repression. Crystallographic studies of unliganded versus liganded nuclear receptors have provided signiWcant insights into the structural and functional relationships of ligandinduced gene activation (Bourguet et al., 1995; Jacobs et al., 2003; Renaud et al., 1995; Rochel et al., 2000) (Fig. 1B). The LBD, domain E, is composed of 12 alpha–helices (H1–H12) that are packed together in a sandwich-like manner, in which H12 contains an AF-2 ligand-dependent activation function. Nuclear receptors function in concert with transcriptional coregulatory proteins, which include basal transcriptional machinery components, chromatin-modifying complexes, corepressors, and coactivators (JaskelioV and Peterson, 2003; Jepsen and Rosenfeld, 2002; Xu et al., 1999). The AF-2 domain is critical for interaction with these various factors. Although certain receptors act as potent transcription repressors in the absence of ligands by binding to corepressors, ligand binding induces several major conformational changes in this region (Brzozowski et al., 1997; Egea et al., 2000; Wagner et al., 1995). In particular, reorientation of H12 upon agonist binding is a prerequisite for dissociation of corepressors and recruitment of coactivators. Conversely, antagonistic ligands are thought to induce a diVerent conformation of the LBD, which prevents the association of coactivators. Therefore, the precise conformation of the LBD and the position of H12 that a nuclear receptor holds upon a speciWc gene promoter largely determines the transcriptional properties of a nuclear receptor. It has become evident that the functions of nuclear receptors can be modulated by several diVerent mechanisms: (1) DNA binding speciWcity and aYnity; (2) nature of the bound ligands (agonists versus antagonists); (3) covalent modiWcations, usually by phosphorylation, in response to speciWc stimuli and environmental cues; and (4) protein–protein interactions occurring between nuclear receptors themselves and among multiple cofactor proteins. When controlling expression of a speciWc target gene, individual mechanisms may function cooperatively to achieve precise regulation. For example, unliganded hormone receptors (e.g., TR, RAR) are coupled to a silenced chromatin state due to recruitment of histone deacetylase complexes by corepressor proteins (Urnov and WolVe, 2001). Conversely, ligand-bound receptors may bind more than one coactivator protein, some of which, such as the cAMP response element binding protein (CREB) binding protein (CBP/p300) and the CBP association factor (p/CAF),

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harbor intrinsic histone acetyltransferase activity. The resultant histone hyperacetylation induces a competent open chromatin configuration, which in turn facilitates the interplay of nuclear receptors with other transcription factors and components of the basal transcription machinery. Thus, it is conceivable that perturbation of any function by nuclear orphan receptors will have a negative impact on the transcriptional control of a target gene. Moreover, the process of orphan nuclear receptor-mediated

TABLE I. Subgroups of Mammalian Nuclear Receptors Class 0

Class I

Class II

Class III

Class IV

SHP (o*)

TR
, 

RXR
, , g (o)

GR

NGFI-B SF-1/ GCNF
, , g FTZ-F1 (o*) (o*)
,  (o*)

DAX-1 (o*) RAR
, , g VDR

Class V

Class VI

COUP-TFs AR (I, II) & EAR2 (o*) HNF4
, , g (o)

PR

PPAR TLX (o*)
, , g (o)

ER
, 

PXR (o)

PNR (o*)

ERR (o)
, , g

CAR & MB67 (o)

TR2 & TR4 (o*)

LXR (o) FXR (o) RevErb
,  (o*) RZR/ROR
, , g (o) O: nuclear orphan receptors; O*: Orphan receptors without identiWed ligand; TR: thyroid hormone receptor; RAR: retinoic acid receptor; VDR: vitamin D receptor; PPAR: peroxisome proliferator-activated receptor; PXR: pregnane X receptor; CAR/MB67: constitutive androstane receptor (previously MB67); LXR: liver X receptor; FXR: farnesoid X receptor; RevErb: reverse ErbA; RZR/ROR: retinoid Z receptor/retinoid acid-related orphan receptor; RXR: retinoid X receptor; COUP-TF: chicken ovalbumin upstream promoter-transcription factor; HNF4: hepatocyte nuclear factor 4; TLX: tailes-related receptor; PNR: photoreceptor-speciWc nuclear receptor; TR2/TR4: testicular receptors 2 and 4; GR: glucocorticoid receptor; AR: androgen receptor; PR: progesterone receptor; ER: estrogen receptor; ERR: estrogen-related receptor; NGFI-B: nerve growth factor-induced nuclear receptor B; SF-1/FTZ-FI: steroidogenic factor-1/ fushi tarazu-factor 1; GCNF: germ cell nuclear factor; SHP: small heterodimeric partner; DAX-1: dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1;
and : alpha and beta, the subtypes of receptors.

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down-regulation is often diverse and complex because multiple modulation mechanisms can be employed simultaneously (Giguere, 1999). The nuclear orphan receptor subfamily is increasing rapidly and, based on molecular phylogeny studies, can be classiWed into seven groups (0 to VI) (Laudet, 1997). Some of the identiWed orphan receptors have been well characterized, but the functions of other candidates remain to be elucidated, particularly in physiologic settings (Table I). Both activation and repression of target gene expression by orphan nuclear receptors are critical for appropriate maintenance of key biologic functions. Furthermore, some orphan receptors exert dual regulatory mechanisms depending on the speciWc target genes that they recognize. This chapter reviews recent progresses in the silencing mechanisms of gene transcription by nuclear orphan receptors chicken ovabulmin upstream promoter-transcription factors (COUP-TFs), dosagesensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1), germ cell nuclear factor (GNCF), short heterodimer partner (SHP), and testicular receptors 2 and 4 (TR2/TR4). These orphan receptors have been well characterized, and they represent four categories within the seven deWned groups of the nuclear receptor subfamily. The repression of gene transcription by these orphan receptors contributes signiWcantly to the control of a wide range of physiologic functions, including development, diVerentiation, and reproduction. Furthermore, in addition to their intrinsic activities, these orphan receptors interfere with steroid and nonsteroid hormonal signaling pathways via interactions with hormone receptors. These orphan receptors’ silencing mechanisms are diverse and complex, which is consistent with the receptors’ multiple functions in regulating distinct sets of target genes.

II. COUP-TFI, II (EAR3, ARP-1), AND EAR2: INHIBITORS OF DIVERSE GENES BY MULTIPLE MECHANISMS Among the rapidly growing numbers of nuclear orphan receptors that have been identiWed, chicken ovalbumin upstream promoter-transcription factor (COUP-TF) is one of the most extensively studied and best characterized transcription factors (Cooney et al., 2001; Tsai and Tsai, 1997; Zhou et al., 2000). Human COUP-TFI was initially identiWed as an activator protein critical for expression of the chicken ovalbumin gene (Ing et al., 1992; Pastorcic et al., 1986; Wang et al., 1989). This receptor was also cloned simultaneously by another group based on its homology to the human erbA gene and termed EAR3 (Miyajima et al., 1988). COUP-TFI AI-regulatory protein-1 (ARP-1) was subsequently isolated by its high homology to COUP-TFI and characterized as a factor that regulates the apolipoprotein AI (ApoAI) gene expression (Ladias and Karathanasis,

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1991). COUP-TF homologues have been cloned in multiple species, including humans, mice, rats, chickens, and Drosophila (Lu et al., 1994; Qiu et al., 1994; Tsai and Tsai, 1997). Sequence analyses revealed that COUP-TFs are typical nuclear receptors and function in the absence of a known ligand (Wang et al., 1989). COUP-TFs are highly conserved across the species (identities greater than 90%) (Fig. 2). Such a strikingly high similarity suggests that the function of COUP-TFs is conserved during evolution. In addition, EAR2, which was cloned at the same time as the EAR3/COUP-TFI gene, is the closest orphan nuclear receptor to COUPTFs within the nuclear receptor family (Miyajima et al., 1988). It shares 86% identity with COUP-TFs at its DBD and negatively regulates the same subsets of target genes. For this reason, EAR2 has often been regarded as a subtype of COUP-TFs’ family members. Although COUP-TFs act predominantly as transcription repressors, they also exert positive regulation on several target genes. One the other hand, EAR2 only exerts negative regulatory actions. COUP-TFs bind preferentially to direct repeats (DR) of the AGGTCA core motif, and they can also recognize inverted (palindromic) or everted repeats of the core sequence with reduced binding aYnity (Cooney et al., 1992). Compared to other nuclear receptors that recognize DR domains with restricted spacing between the two half-sites, COUP-TFs exhibit substantial binding flexibility where they bind as homodimers to a variety of DR elements with diVerent lengths of spacers. COUP-TFs’ binding aYnity is highest for the DR1 element and decreases in the order of DR6>DR4>DR8>DR0>DR11 for other DR motifs (Cooney et al., 1992; Kadowaki et al., 1992). Consistent with this, characteristic COUP-TFs bind to diVerent DR or palindromic response elements that are recognized by the following nuclear receptors:

FIGURE 2. Functional domains of COUP-TFs. COUP-TFI/EAR3 and COUP-TFII/ ARP-1 are highly homologous. The LBD of COUP-TFs harbors multiple functions, including active repression (solid line with everted arrow heads), transrepression (dashed dots), and the interaction surface for corepressors NCoR and SMRT (Wlled rectangular box). A region containing the most C-terminal 15 amino acids also harbors an activation function (Wlled diamond). COUP-TFI interacts with Sp1 through its DBD, while its interaction with MyoD requires the DBD, the hinge region, and a small stretch of sequences at the N-terminal region of the LBD.

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RAR, RXR, TR (Cooney et al., 1992), peroxisome proliferator-activated receptor (PPAR) (Marcus et al., 1996; Nishiyama et al., 1998), hepatocyte nuclear factor 4 (HNF4) (Mietus-Snyder et al., 1992; Yanai et al., 1999), and estrogen receptor (ER) (Liu et al., 1993). COUP-TFs consequently antagonize the hormone-induced target gene transactivation. In addition, COUP-TFs inhibit basal promoter activities for a number of genes. They repress the genes for apolipoproteins (Ladias et al., 1992), c-mos protooncogene (Lin et al., 1999), and Oct4 (Schoorlemmer et al., 1994) via DR1 domains. COUP-TFs also repress rat insulin 2 (Hwung et al., 1988), arrestin (Lu et al., 1994), and HIV-LTR genes (Cooney et al., 1991) by binding to a DR6, DR7, or DR9 element, respectively. COUP-TFs and EAR2 cause potent repression of the luteinizing hormone receptor (LHR) gene (Zhang and Dufau, 2000, 2001), the oxytocin gene (Chu and Zingg, 1997), and a neurotransmitter gene, GRIK5 (Chew et al., 1999). They also inhibit the acyl-CoA dehydrogenase gene promoter at everted repeats separated by 8 and 14 nucleotides (Carter et al., 1994). Moreover, gene-targeting studies have demonstrated that COUP-TFs play critical roles in neurogenesis, angiogenesis, and heart development. COUP-TFI null mice die shortly after birth from starvation and dehydration (Qiu et al., 1997). Death is caused by defects in the development of the peripheral nervous system, where swallowing and suckling functions are compromised. Disruption of the COUP-TFII gene is lethal at the embryonic stage because of defects in angiogenesis, vascular remodeling, and heart development (Pereira et al., 1999). COUP-TFs induce gene silencing through several mechanisms, including competition for binding to DNA cis-elements, quenching through titrating out RXR, and active repression and transrepression (Leng et al., 1996) (Figs. 3 and 4). In the presence of their cognate hormones, vitamin D3 receptor (VDR) RAR, or TR activate target gene expression through binding to DR3, DR4, or DR5 hormone response elements, respectively. The promiscuous binding of COUP-TFs to DR domains thus provides a molecular mechanism in which their competitive occupancy of the same DNA elements inhibits the gene transactivation induced by these hormone receptors (Ben-Shushan et al., 1995; Cooney et al., 1992; Stephanou et al., 1996; Tran et al., 1992) (Fig. 3, IA). In addition, binding of EAR2 to the retinoic acid response element (RARE) motif suppresses both basal and RA-induced mouse renin gene promoter activity (Liu et al., 2003). Repression by COUP-TFs of the PPAR-induced gene activation and HNF4 function in the control of many liver-speciWc genes is attributed to a similar mechanism. In the regulation of the estrogen responsive lactoferrin gene, COUP-TFs down-regulate the estrogen receptor activation through an overlapping COUP/ER binding module (Liu et al., 1993). Their occupancy of this composite site blocks the binding of ER, which is followed by the loss of estrogen response of these target genes. This mechanism also applies to

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FIGURE 3. Models for passive and transrepression of target gene expression by COUPTFs. (I) The COUP-TFs-elicited passive gene repression is achieved by competing with nuclear receptors (RAR, TR, VDR, PPAR, and HNF4) for occupancy of the same DR elements (A) or by titrating out RXR via protein–protein interaction between COUP-TF and RXR (B). Disruption of the formation of active heterodimer between RAR, TR, VDR, or PPAR and RXR or of RXR homodimer results in gene silencing (B). (II) Transrepression by COUP-TFs is independent of their binding to the target gene promoter regions, whereas the repression is achieved through competition of COUP-TFs with the transcription activator proteins (e.g., MyoD, HNF4) for the same sets of coactivators (e.g., p300 or SRC-1) via protein–protein interactions. PIC, preinitiation complex. COUP-TFs’ repressed SF-1 target gene activation, during which the SF-1 site is embedded in a COUP-TF binding element (Wehrenberg et al., 1994; Xing et al., 2002). The notion that heterodimeric association of VDR, RAR, TR, or PPAR with RXR is a prerequisite for these hormone receptors to achieve higheYciency binding to their cognate target sites has identiWed RXR as a generic partner critical in these hormone signaling pathways (Zhang et al., 1992). COUP-TFs, in addition to their homodimeric binding activity, can heterodimerize with RXR in the presence of DNA binding elements. Such formation of an inactive COUP-TF:RXR dimer titrates out RXR for binding to RAR or other hormone receptors (e.g., VDR, TR) and thereby alleviates the hormone-induced gene expression (Kliewer et al., 1992; Widom et al., 1992) (Fig. 3, IB). Overexpression of RXR relieved the squelching eVect of COUP-TFs, conWrming that quenching of the RXR receptor resulted in the inhibition. Moreover, the genes that are activated by

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FIGURE 4. Models for active silencing of target gene expression by COUP-TFs. (A) Repression of human LHR gene expression by COUP-TFI/EAR3 from cross-talk among Sp1/Sp3, COUP-TFI/EAR3, and TFIIB. COUP-TFI/EAR3 bound to the DR0 motif interacts with Sp1/Sp3 bound to the Sp1(I) site. Such interaction prevents the association of TFIIB to the Sp1 (I) site without aVecting the recruitment of TFIIB to the hLHR gene core promoter region. Anchoring of TFIIB at the Sp1(I) site is independent of prior binding of COUPTFI/EAR3 to the DR0 element, and the interaction of TFIIB with Sp1/Sp3 is indirectly bridged by a currently unidentiWed protein(s), putative tethering protein (PTP). The COUP-TFI/EAR3reduced association of TFIIB to the Sp1/Sp3–DNA complex may induce a nonproductive or less productive form of preinitiation complex (PIC), where the recruitment of RNA Pol II to the hLHR promoter is decreased in JAR cells. This occurs when the hLHR gene expression is subjected to a repressed state by COUP-TFI/EAR3. TSS stands for the transcriptional start site (B) Alternative mechanism of active repression by COUP-TFs depends on speciWc binding of COUP-TFs to their cognate response elements in the target gene promoters. Corepressor proteins SMRT/NCoR that are recruited to COUP-TFs through protein–protein interaction act as bridging molecules to exert a negative impact on the PIC complex. In addition HDAC/Sin3 complexes may associate with COUP-TFs by direct interaction with COUP-TFs or by association with NCoR/SMRT. The resultant compressed/closed chromatin structure induced by histone hypoacetylation contributes to the target gene silencing (Zhang and Dufaul (2003a). Reprinted with permission from Mol. Cell. Biol.) RXR:RXR homodimer in presence of 9-cis retinoid acid are suppressed by COUP-TFs through their association with RXR. Therefore, competition for hormone response elements and for RXR are dual parallel mechanisms for COUP-TFs to suppress the response of the target genes to vitamin D3 T3, retinoid acid, or PPARs. COUP-TFs also possess intrinsic repression activity, which causes active repression of several target genes (Fig. 4B). The active repression domain is located in LBD (Achatz et al., 1997), which interacts with corepressor proteins nuclear receptor corepressor (NCoR) and its variant RIP13deltal as well as silencing mediator for retinoid acid and thyroid hormone receptors (SMART) that potentiate COUP-TFs’ silencing eVect (Bailey et al., 1997; Shibata et al., 1997). Such active repression by COUP-TFs

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also involves participation of histone-modiWcation complexes with interactions between COUP-TFs and histone deacetylase (HDAC) (Smirnov et al., 2000) or between NCoR or SMRT and HDAC1/mSin3 corepressor complex (Alland et al., 1997; Nagy et al., 1997). SpeciWc inhibition of HDAC activity by trichostatin A (TSA) abolishes the silencing eVect, which further indicates that the condensed chromatin structure caused by histone hypoacetylation may provide an appropriate environment, favoring or stabilizing the recruitment of corepressor proteins by COUP-TFs. In addition, two COUPTF interacting proteins CIP1 and CIP2 have been isolated through yeast twohybrid screening of an adenoma cDNA library (Kobayashi et al., 2002). Coexpression of CIP1 or CIP2 with COUP-TFs enhanced the COUP-TFs’ mediated repression of several steroidogenic genes, including CYP17 and CYP11B2 (Kobayashi et al., 2002). Predominant expression of CIP proteins in steroidogenic tissues such as testes, ovaries, and adrenal glands, where COUP-TFs are coexpressed, indicates that CIP1 and CIP2 function as corepressor proteins for COUP-TFs in the control of steroidogenesis. Moreover, COUP-TFII and EAR2 heterodimer displayed high but distinct binding speciWcity compared to the respective homodimers of each receptor (Avram et al., 1999). The fact that EAR2 transcript expression overlaps precisely with COUP-TFII expression in several mouse tissues and embryonic carcinoma cell lines suggests that heterodimeric interaction between COUPTFII and EAR2 may deWne an alternative mechanism for the function of these orphan receptors. COUP-TFI and EAR2 cause marked repression of the LHR gene promoter activity in a dose-dependent manner. The repression is mediated by the binding of these receptors to an imperfect DR0 motif in the LHR gene promoter, while mutation of the DR0 domain abolishes the silencing eVect (Zhang and Dufau, 2000, 2001). The Wnding that the inhibition is independent of changes in histone acetylation levels suggests a mechanism other than involvement of corepressor/HDAC activity in the negative modulation of the LHR gene expression (Zhang and Dufau, 2002, 2003b). Recent studies have revealed a novel mechanism for active repression of the LHR gene by COUPTFs caused by cross-talk among COUP-TFI, Sp1/Sp3, and basal transcription factor TFIIB (Zhang and Dufau, 2003a) (Fig. 4A). The repression depends on a proximal Sp1/Sp3 binding site, designated as Sp1(I) site, which is one of the two essential Sp1 sites in the control of the basal promoter activity. Both Sp1 and Sp3 are required for the inhibition, and mutation of the Sp1(I) site to disrupt the Sp1/Sp3 binding abolishes the COUP-TFI silencing eVect. The functional cooperation between the DR0 and the Sp1 domain is supported by mutual recruitment of COUP-TFI and Sp1/Sp3 bound to their cognate sites. COUP-TFI interacts with Sp1 through its DBD but also requires its N-terminal region, and deletion of these two domains greatly compromises the repression. Furthermore, TFIIB interacts with Sp1(I)bound Sp1/Sp3 in addition to its association with COUP-TFI and the

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TATA-less human LHR (hLHR) gene core promoter region. Such interaction is indirect, relying on adaptor protein(s) present in the nuclear extracts of human placental choriocarcinoma JAR cells. COUP-TFI speciWcally decreases the association of TFIIB to the Sp1(I) site without interfering with its interaction with the core promoter. The C-terminal region of COUP-TFI, which does not participate in its interaction with Sp1, is required for its inhibitory function and may aVect the association of TFIIB with Sp1. The COUP-TFI-elicited disassociation of TFIIB with Sp1 is also reflected in the reduced recruitment of RNA PoLII to the hLHR gene promoter. Overexpression of TFIIB counteracts the inhibition by COUP-TFI and activates hLHR gene transcription in an Sp1(I) site-dependent manner. Taken together, these Wndings indicate that TFIIB is a key component in the regulatory control of COUP-TFI and Sp1/Sp3 on the initiation complex, where repression of the hLHR gene transcription by COUP-TFI results from its perturbation of communication between Sp1/Sp3 at the Sp1(I) site and the basal transcription initiator complex. Thus, the control of the TATA-less hLHR gene by active repression employs a novel mechanism that is diVerent from those operative for most of the TATA-box genes. COUP-TFs transrepress the expression of target genes by a mechanism that is independent of their cognate DNA elements (Fig. 3, II). In the case of transactivation of the human apo-B gene by HNF4, HNF3
, or C/EBP, COUP-TFII markedly antagonizes the individual transactivator’s function without binding to the apo-B gene promoter (Achatz et al., 1997). The domain that harbors such a transrepression function is located in the COUP-TFII DNA binding region and also in a segment spanning amino acid residues 193 to 399 but does not include its repressive domain. Tethering of COUP-TFs to TR or RAR via protein–protein interactions between their respective LBDs also results in potent transrepression of TR- or RAR-activated gene expression (Berrodin et al., 1992; Butler and Parker, 1995). In addition, COUP-TFII inhibits myogenesis through repression of MyoD-dependent transcription in the absence of its binding site (Bailey et al., 1998). Such repression was mediated through association of COUP-TFII’s DBD and the hinge region with the N-terminal domain of MyoD. Overexpression of coactivator p300 relieves the inhibition by COUP-TFII, indicating cross-talk between COUP-TFII, MyoD, and p300. COUP-TFII, MyoD, and p300 interact in a competitive manner. Increasing the amount of COUP-TFII reduces the association of MyoD with p300 and elicits an inhibitory regulation of MyoD-induced myogenic diVerentiation. Moreover, EAR2 directly interacts with and decreases thyroid hormone receptor TR1 binding activity to its target genes. EAR2 represses both the 3,3,5-triiodo-L-thyronine (T3) induced and T3-independent TR1 gene activation in a cell-speciWc manner. Such repression is reversed by the coactivator SRC-1, indicating that the balance of corepressor (e.g., EAR2) and coactivator actions has an important role in the control of the TR-mediated responses (Zhu et al., 2000).

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Up-regulation of target gene expression by COUP-TFs is also mediated through several mechanisms. One involves protein–protein interactions of COUP-TFs with other transcription factors, whereas the binding of COUPTFs to the response elements are either disposable or absent. COUP-TFI exhibits strong activation of HIV long terminal repeat and NGFI-A genes through a Sp1 binding site and up-regulates vHNF gene promoter activity through an octomer protein-binding site element (Pipaon et al., 1999; Power and Cereghini, 1996; Rohr et al., 1997). Interaction of COUPTFI with Sp1 or Oct1 is dependent on COUP-TFI’s DBD, while the C-terminal 15 amino acids are also crucial for the transactivating function. The extreme C-terminal region of COUP-TFI is responsible for the recruitment of a coactivator. Steroid receptor coactivator-1 (SRC-1), glucocorticoid receptor interacting protein 1 (GRIP1), and p300 interact with COUP-TF and potentiate its activation of HIV and NGFI-A genes. In addition, COUPTFs act as an accessory factor for induction of phosphoenolpyruvate carboxykinase (PEPCK) gene in response to glucocorticoids (Hall et al., 1995; Scott et al., 1996). Although the eVect of glucocorticoids is mediated by a glucocorticoid receptor (GR) binding to two glucocorticoid response elements (GRE), the maximal induction depends on two COUP-TF binding sites that flank the GRE domains. Although COUP-TFs binding to these sites has no eVect on the basal promoter activity, they work synergistically with GR to elicit marked transactivation. In addition, COUP-TFI is required for induction of the RAR gene in the presence of retinoic acid, which contributes to the RA-induced growth inhibition and apoptosis in cancer cells (Lin et al., 2000). In this case, speciWc binding of COUP-TFI to a DR8 motif in the RAR promoter enhanced the RAR
:RXR transactivation by increasing recruitment of coactivator, cyclic AMP response element-binding (CREB) protein to RAR
. Taken together, multiple mechanisms have been derived for COUP-TFs-regulated gene expression, in which positive or negative regulation is primarily determined by the speciWc target genes recognized. Such diversity and complexity is thus consistent with the multiple functions of COUP-TFs in the control of diVerent target genes.

III. DAX-1: SILENCING IN THE CONTROL OF STEROIDOGENIC AND SEX-DETERMINING TARGET GENES DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1) is a member of the nuclear receptor superfamily with major action in development of steroidogenic and reproductive tissues (Lalli and Sassone-Corsi, 2003; Meeks et al., 2003). DAX-1 mutations cause the X-linked form of adrenal hypoplasia congenita (AHC) that is invariably associated with hypogonadotropic hypogonadism

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(HHG) (Muscatelli et al., 1994; Tabarin, 2001). Duplication of the DAX-1 gene locus on chromosome Xp21 causes male to female sex reversal in XY individuals with a normal SRY (male sex determinant) gene (Bardoni et al., 1994). Consistent with its critical role in adrenal and gonadal development and diVerentiation, expression of the DAX-1 gene is predominantly observed in the adrenal cortex, testicle, ovary, hypothalamus, and anterior pituitary (Guo et al., 1995; Swain et al., 1996). The DAX-1 gene consists of two exons separated by an intron that encodes a protein of 470 amino acids (Burris et al., 1996). DAX-1 is an atypical nuclear receptor because it does not contain a canonical zinc Wnger DBD characteristic of other members of the nuclear receptor superfamily (Fig. 5). Instead, the Nterminal of the DAX-1 gene is composed of three- and -a-half repeats of 65 to 67 amino acids. The C-terminal region of the DAX-1 gene harbors a putative LBD that resembles the ligand binding region of other nuclear receptors. In particular, its helix 12 (H12) region contains a conserved XE motif (: a hydrophobic amino acid; X: a nonconserved amino acid) that in other nuclear receptors mediates ligand-inducible transcriptional activation. However, no ligand for this orphan receptor has yet been identiWed. The unique N-terminal of DAX-1 confers novel binding activity by which this orphan receptor recognizes its target genes (Fig. 5). DAX-1 binds to a DNA hairpin structure in the promoter of the steroidogenic acute regulatory protein (StAR) gene and potently represses its transcription

FIGURE 5. Structure and function relationship in DAX-1-mediated regulation of target gene expression. The N-terminal region of DAX-1 contains three repeats of leucine-rich motifs as indicated by LYNML. DAX-1 binds to DNA hairpin loop structure and RNA in the polyribosome complexes through the leucine-repeat motifs. This region also mediates its interaction with SF-1, ER
, LRH-1, ERR, and AR receptors, in which competitive binding with coactivators for AF-2 domain of the respective nuclear receptor causes target gene silencing. The C-terminal LBD region of DAX-1 harbors intrinsic repressive activity and also participates cooperatively in the RNA binding activity. Nuclear corepressors NCoR and Alien interact with this region to potentiate the DAX-1’s inhibitory function. H12 is critical for the nuclear localization of DAX-1.

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(Zazopoulos et al., 1997). The StAR gene encodes a protein that mediates transport of cholesterol from the outer to the inner mitochondrial membrane, where it is converted to pregnenolone by the cytochrome P450 cholesterol-side chain cleavage enzyme) (CYP11A1) during steroid biosynthesis (Stocco, 2001). Consequently, DAX-1-blocked StAR gene transcription is followed by a marked reduction of steroidogenesis. The silencing eVect of DAX-1 on StAR gene transcription is attributed to an intrinsic repression function located in the DAX-1 C-terminal LBD (Ito et al., 1997). Such DAX-1-mediated inhibition is both cell-type and promoter speciWc. Furthermore, DAX-1 is also an eYcient RNA binding protein and associates with mRNA in polyribosome complexes (Lalli et al., 2000). In this case, the N-terminal repeat sequences and the C-terminal domain of DAX-1 are both important since the two domains function cooperatively to achieve competent RNA binding. Moreover, the DNA/RNA binding activities of DAX-1 are compromised by mutations present in patients with hypoplasia, suggesting that DAX-1 exerts its regulation on target gene expression at both transcriptional and posttranscriptional levels under certain physiologic conditions. The observation that the spatial and temporal expression of DAX-1 is closely related to that of another orphan receptor, SF-1, suggested that these receptors might work in concert to modulate endocrine functions (Ikeda et al., 1996, 2001; Swain et al., 1996). SF-1 has been directly implicated in the regulation of Mu¨ llerian-inhibiting substance (MIS) gene expression and also in the transcriptional regulation of steroid hydroxylase and aromatase genes (Giuili et al., 1997; Parker, 1998). Mice harboring a SF-1 null mutation display adrenal and gonadal agenesis, loss of pituitary gonadotropins, and altered structural properties of the ventromedial hypothalamus (Ingraham et al., 1994; Luo et al., 1994). The close resemblance of the phenotype by SF-1 disruption to that of the DAX-1 mutation-induced human hypoplasia congenital symptoms supports a functional correlation between these two orphan receptors in adrenal and reproductive development and function (Achermann et al., 2001b; Beuschlein et al., 2002). DAX-1 strongly represses the SF-1-mediated reporter gene activation through its direct interaction with SF-1; such inhibition does not aVect the SF-1 binding activity to its response element (Ito et al., 1997). Although the DAX-1 N-terminal region is responsible for its interaction with SF-1, its inhibitory function is attributed to its C-terminal LBD. To date, more than 60 diVerent DAX-1 mutations have been found in patients with X-link AHC disease (Phelan and McCabe, 2001). Most are frameshift or nonsense mutations that result in premature truncation/deletion of the DAX-1 protein. Relatively few missense mutations have also been detected, and these mutations appear to cluster in certain regions of the C-terminal of DAX-1. Hence, the loss of DAX-1-mediated negative regulation of the SF-1

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gene due to mutations (deletion or point mutations) at the C-terminal inhibitory domain may contribute to AHC disease (Lalli et al., 1997). Consistent with the inhibitory role of DAX-1 in SF-1-modulated target gene expression, the expression of the aromatase CYPl9 gene, which is under control of SF-1, is signiWcantly elevated in DAX-1 deWcient male mice (Wang et al., 2001). So far, the target genes that are negatively regulated by DAX-1 in this manner also include genes for CYP11A1, 3 beta-hydroxysteroid dehydrogenase (3-HSD) (Hu et al., 2001; Lalli et al., 1998), CYP17 (Hanley et al., 2001), the relaxin-like factor (Koskimies et al., 2002), inhibin alpha (Achermann et al., 2001a), high-density lipoprotein (HDL) receptor (Lopez et al., 2001), and others. It is has been proposed that silencing of expression of diVerentiation genes (e.g., StAR, steroid hydroxylases) by DAX-1 in the adrenal cortex may be a prerequisite for proliferation of the deWnitive adrenocortical zone in the critical postnatal period for humans In the absence of repressive function of DAX-1, abnormal early expression of steroidogenic genes occurs in the deWnitive zone, and consequently cell proliferation, diVerentiation, and zonation are disrupted. Adrenal hypoplasia would then follow the physiologic regression of the fetal zone (Lalli, 2003). DAX-1 can also interfere with SF-1’s interactions with other transcriptional factors. It is known that expression of the MIS gene is cooperatively regulated by actions of several transcriptional factors, including Sox9, SF-1, Wilms’ tumor 1 (WTI) GATA binding protein 4 (GATA-4), and DAX-1 (Arango et al., 1999; Nachtigal et al., 1998; Shimamura et al., 1997). Appropriate spatial-temporal expression of the MIS gene is critical for mammalian sex diVerentiation because the MIS hormone produced by Sertoli cells mediates regression of the Mu¨ llerian ducts in males during testes diVerentiation (MacLaughlin and Donahoe, 2002). WT1 or GATA-4 synergize with SF-1 to promote MIS gene expression in Sertoli cells. In contrast, DAX-1 causes strong repression of the SF-1-induced gene activation by disruption of synergism between SF-1 and WT1 or GATA-4 (Nachtigal et al., 1998; Tremblay and Viger, 2001). Such inhibitory eVect is mediated by direct protein–protein interaction of DAX-1 with the DNA-bound SF-1. Moreover, targeted disruption of the DAX-1 gene in mice reveals that DAX-1 is also essential for the maintenance of spermatogenesis in males (Yu et al., 1998). The knockout animals display progressive degeneration of the testicular germinal epithelium, which causes sterility in males. In contrast, the loss of DAX-1 function in females does not aVect ovarian development or fertility, indicating that DAX-1 is not an ovarian determining gene. Transgenic studies further demonstrate that XY mice carrying extra copies of the DAX-1 gene antogonizes Sry action in mammalian sex determination, and overexpression of DAX-1 causes sex reversal (Swain et al., 1998). These Wndings conWrm that DAX-1 is responsible for dosage-sensitive sex (DSS) reversal as initially identiWed in human DSS reversal patients.

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The corepressors NCoR and Alien but not SMRT (Crawford et al., 1998) participate in the silencing mechanism of DAX-1 (Fig. 5). Recruitment of such corepressor proteins by DAX-1 suggests a novel regulatory mechanism by which corepressors can be tethered to a transactivator protein such as SF-1, in addition to the widely-held view that corepressor proteins usually associate with transcriptional repressors. It has been shown that natural AHC mutations of DAX-1 markedly diminish the recruitment function of DAX-1 for both NCoR and Alien, signiWcantly impairing the silencing eVect of DAX-1 (Altincicek et al., 2000). The abolishment of corepressor binding activities of DAX-1 due to its mutations in patients with AHC may contribute to the pathogenesis of this disease. More recent Wndings have shed further light on the molecular mechanism of the DAX-1 and SF-1 interactions. The N-terminal domain of DAX-1 contains repeated leucinerich LXXLL-related sequences, a motif that usually occurs in nuclear receptor coactivator proteins and mediates their interaction with nuclear hormone receptors (Suzuki et al., 2003). This leucine-rich motif determines the interactions of DAX-1 with SF-1 and several other nuclear receptors, including estrogen receptor
(ER
), liver receptor homologue-1 (LRH-1) estrogen-related receptor 2 (ERR2), and fly fushi tarazu factor 1. Thus, it is possible that the N-terminal LXXLL-repeated motifs of DAX-1 serve as an anchor site to tether DAX-1 to SF-1 and that the C-terminal intrinsic repression domain inhibits SF-1’s transactivating function through recruitment of corepressor proteins (NCoR and Alien). On the other hand, the silencing eVect of DAX-1 on SF-1 action is not limited to SF-1 activated genes but also extends to gene(s) potentially inhibited by SF-1. In the case of the human CYP11B2 gene, which encodes an enzyme that is critical for aldosterone production in the adrenal gland, coexpression of DAX-1 and SF-1 overcomes SF-1-repressed gene transcription in transient transfection studies (Bassett et al., 2003). These results demonstrate the diVerence in the control of the human CYP11B2 gene expression from other genes that are up-regulated by SF-1 during steroidogenesis. DAX-1 has also been found to participate in hormonal signaling pathways through cross-talk with nuclear hormone receptors. DAX-1 interacts with the ligand-activated estrogen receptors alpha and beta (ER
and ER) through DAX-1 N-terminal LXXLL-related motifs and signiWcantly reduces ER activation (Zhang et al., 2000). Since DAX-1 shares a similar interaction domain with coactivator proteins in the recognition of nuclear hormone receptors, by occupying the LBD of ERs, DAX-1 may block or mask recruitment of coactivators to ERs and, therefore, have a negative impact on ER-induced gene transcription. It is also possible that subsequent recruitment of some corepressors by DAX-1 upon binding to ERs may contribute to the down-regulation. Furthermore, identiWcation of the androgen receptor (AR) as a novel target of DAX-1-mediated repression

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provides further insights into the critical role of DAX-1 in male reproduction in vivo because DAX-1 (/) mice have defects in spermatogenesis (Holter et al., 2002; Yu et al., 1998). Studies showed that DAX-1 also interacts with AR and markedly inhibits ligand-dependent AR transactivation activities. Direct physical interaction was observed between these two receptors, and the interaction domains are mapped to the Nterminal of DAX-1 and the ligand binding AF-2 activation domain of AR. In addition, besides its intrinsic silencing function, DAX-1 exerts a novel mechanism in regulation of AR action by tethering AR to cytoplasm. Therefore, the altered cellular localization of AR causes an impaired AR function. The physiologic relevance of DAX-1-mediated repression of AR is supported by evidence that expression of DAX-1 is also observed in the human prostate, an important target site of androgen action. In particular, expression of DAX-1 was detected mainly in epithelial cells, where high levels of AR and ER were expressed. In addition to the observation that the DAX-1 AHC mutants exhibited impaired activities in corepressor recruitment and silencing eVect, the subcellular localization of the DAX-1 AHC mutant proteins was invariably shifted to the cytoplasm, independent of an intact nuclear localization signal (NLS) present at DAX-1’s N-terminal (Lehmann et al., 2002). The altered localization is also evident for the GAL4 DBD with its own NLS fused to the mutant DAX-1 C-terminal region, indicating that the nuclearto-cytoplasmic shift is solely attributed to the mutations of the DAX-1 Cterminal region. The cytoplasmic localization of DAX-1 AHC mutants inversely correlates with their transcription repressive activities, and the H12 region is identiWed as critical for maintaining DAX-1 as a nuclear protein. Consistent with these Wndings, a DAX-1 mutant found in a patient with late adult onset of adrenal insuYciency and incomplete HHG exhibited least shifting eVect on DAX-1 localization. Recent studies further show that the nuclear-to-cytoplasmic shift of the DAX-1 AHC mutants is likely caused by protein misfolding because the DAX-1 mutants are much more sensitive to proteolysis when compared to the wild-type DAX-1 (Lehmann et al., 2003). It is therefore suggested that the altered conformation present in the C-terminal mutant DAX-1 may induce formation of a novel interacting surface for putative cytoplasmic anchoring site(s), consequently causing the shift of DAX-1 from nucleus to cytoplasm. Taken together, the current understandings have revealed that DAX-1, an atypical nuclear orphan receptor, functions as a potent repressor in adrenal and reproductive axis development and function through perturbation of targeted gene expression modulated by StAR, SF-1, ER, and AR. DAX-1 also possesses unique DNA/RNA binding activities. More than one mechanism exists for DAX-1-mediated gene silencing, in which combined eVects of active repression, recruitment of a corepressor, mask of coactivator binding, and alteration of a hormone receptor’s localization

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may work together to achieve a precisely programmed biologic function. These various studies, together with the Wndings that all DAX-1 AHC mutants display cytoplasmic-oriented relocalization, have provided a molecular basis for DAX-1 defect-induced adrenal and gonadal disorders characterized by naturally occurring DAX-1 mutations that abolish or signiWcantly impair DAX-1 function as a transcriptional repressor.

IV. GCNF: NEGATIVE CONTROL DURING GAMETOGENESIS AND EMBRYONIC DEVELOPMENT Germ cell nuclear factor (GCNF), which is also known as retinoid acid receptor-related testis-associated receptor (RTR) and neuronal cell nuclear factor (NCNF), is an orphan member of the nuclear receptor superfamily; no ligand has been identiWed for its action. The GCNF gene was initially isolated from a mouse cDNA library and later was identiWed in human and Xenopus laevis (Chen et al., 1994; Hirose et al., 1995b; Joos et al., 1996; Susens and Borgmeyer, 1996). The mouse GCNF (mGCNF) gene is composed of 11 exons, 2 that encode a protein of 495 amino acids. The human GCNF (hGCNF) contains 476 amino acids, and its gene has been located on chromosome 9 at locus q33–34 (Agoulnik et al., 1998). Sequence alignment of GCNF genes isolated from diVerent species has demonstrated that the genes are highly homologous and evolutionary conserved (Greschik and Schule, 1998). In addition, GCNF genes from humans and Xenopus laevis harbor deletions at their N-termini when compared to that of mouse GCNF, but it is yet unclear if these diVerences exert any species-speciWc impact on the function of GCNF. Furthermore, GCNF contains typical structural characteristics of the nuclear receptor superfamily, including a DNA binding domain (DBD) and a putative ligand binding domain (LBD) (Fig. 6). Because of the lack of the AF-2 transcriptional activation function in H12 of GCNF’s LBD, this orphan receptor was proposed to function as a transcriptional repressor (Susens et al., 1997). Sequence analyses show that GCNF does not display substantial homology to any other nuclear receptors and that it is the only member of group VI of the nuclear receptor family (Giguere, 1999). The closest homologue of GCNF is the retinoid X receptor (RXR), with which it shares 32 to 34% overall amino acid identity and 61% identity at the DBD. Temporal and tissue-speciWc expression of GCNF has been intensively studied in humans, rodents, and Xenopus laevis. In addition, its binding properties were investigated and the response elements have been identiWed. SigniWcant evidence about the physiologic role of GCNF during early embryonic development has been derived from gene-targeting studies using

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FIGURE 6. DNA binding and inhibitory properties of GCNF. GCNF binds as homodimer to AGGTCA direct-repeat motifs with zero spacing (DR0) with or without a 50 extension sequence (Wlled square box). GCNF also recognizes an extended AGGTCA half-site as homodimer, where dimerization involves H3 and H12 as novel association domains. GCNF lacks AF-2 domain but possesses intrinsic repression activity in its C-terminal LBD region (hatched region). Recruitment of corepressor NCoR potentiates the GCNF-induced gene silencing, and the amino acids represented by vertical bars are critical for GCNF to interact with NCoR. GCNF inhibits expression of Prm1, Prm2, Oct4, BMP-15, and GDF-9 genes through binding to DR0 motifs within these gene promoters.

animal models; the studies reveal that GCNF is essential for embryonic survival and development (Hummelke and Cooney, 2001). Other studies have demonstrated that GCNF silences transcription of genes for protamines 1 and 2 as well as Oct4. GCNF also transrepresses the gene transcription modulated by the estrogen related-receptor alpha 1 (ERR
1). The name of GCNF originated from the evidence that GCNF expression is predominant in germ cells of testes and ovaries of adult mammals. The highest GCNF expression in the mouse is observed in round spermatids at stages VII and VIII and less in spermatocytes (Zhang et al., 1998). Moreover, mouse GCNF (mGCNF) mRNA is not detected in hypogonadal mouse testes where the development of spermatogenic cells stops after the Wrst prophase of meiosis (Mason et al., 1986). In contrast to the mouse, GCNF expression in human testes is predominant in spermatocytes but less in round spermatids (Agoulnik et al., 1998). The cell-speciWc expression of GCNF in spermatogenic cells of adult male tests indicates that it may play a regulatory role during terminal diVerentiation of spermatogenic cells, in particular in spermatids prior to the initiation of nuclear elongation and condensation. GCNF expression also has been observed in growing ovarian follicles but not in primordial oocytes, and its expression persists in the ovulatory follicles (Katz et al., 1997; Lan et al., 2003a). GCNF is also present in ovulated oocytes and preimplantation embryos, indicating that

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GCNF may play a maternal role in zygotic development prior to implantation (Lan et al., 2003a). Recent studies have shown that mice with an oocyte-speciWc GCNF gene knockout display impaired fertility and prolonged diestrus resulting from reduced steroid hormone levels (testosterone, estradiol, and progesterone). In addition, abnormal double-oocyte follicles are observed in GCNF/ female mice. Mechanistically, disruption of GCNF expression abrogates the GCNF-mediated silencing of BMP-15 and GDP-9 gene transcription at diestrus. The aberrant steroidogenesis with reduction of StAR, 3-HSD, and 17
-hydroxylase gene expression in the somatic cells of the ovary result from overexpression of the BMP-15 and GDP-9 genes in oocytes at diestrus. The fact that BMP-15 and GDF-9 are important components of the paracrine signaling pathway in the ovary indicates that GCNF aVects female fertility by regulating paracrine communication between oocyte and somatic steroidogenic cells via repression of the BMP-15 and GDF-9 gene expression (Lan et al., 2003b). Expression of GCNF is widely detected in mouse embryos after gastrulation and later mainly exists in the developing nervous system (Bauer et al., 1997; Susens et al., 1997). Strong expression of GCNF is also shown in embryonic carcinoma cells (Lei et al., 1997). Mouse embryos harboring germ line-targeted mutation of GCNF died around 10.5 days postcoitum (E10.5) because of cardiovascular complication (Lan et al., 2002). Prior to death, signiWcant developmental defects were observed in GCNF/ mice; these defects included posterior truncation, ectopic tail-bud formation, unclosed neural tube, compromised somitogenesis, and other abnormalities. In addition, studies of Xenopus embryos indicate that the xGCNF gene is expressed between the gastrula and mid-neurula stages. Depletion of embryonic xGCNF expression shows that xGCNF function is required for morphogenetic cell movement during neurulation (Barreto et al., 2003). Characterization of DNA binding activity of GCNF has revealed unique properties for this orphan receptor as a sequence-speciWc DNA binding protein. GCNF binds speciWcally and with high aYnity as homodimer to a direct-repeat (DR) motif of AGGTCA half-site with zero spacing (DR0) (Cooney et al., 1998; Yan et al., 1997). GCNF can also bind strongly as a homodimer to a consensus SF-1 response element, thus displaying a novel dimeric binding property for an extended half-site (Greschik et al., 1999). Monomeric binding of GCNF to the SF-1 site with markedly lower activity has also been observed. Homodimeric binding of GCNF to an extended half-site requires a novel dimerization function present in its DBD and the participation of helix 3 (H3) and H12 of its putative LBD. IdentiWcation of H3 and H12 as critical for dimerization is unique for GCNF since dimerization of other receptors depends on either helices 9 and 10 or helices 5 through 7. Moreover, GCNF does not bind direct-repeat sequences separated by one to six nucleotides (DR1–6) (Cooney et al., 1998). In contrast to other

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nuclear orphan receptors, such as COUP-TFs that display flexibility in recognition of DR motifs with varied spacing lengths (Tsai and Tsai, 1997), GCNF is the Wrst nuclear receptor identiWed that solely binds to DR0 motifs. Moreover, unlike the thyroid hormone receptor (TR) or retinoid acid receptor (RAR) that heterodimerize with RXR to achieve high aYnity bindings to DR elements, no heterodimerization is observed between GCNF and RXR (Borgmeyer, 1997). GCNF represses activities of heterologous and natural gene promoters (protamines 1 and 2 and Oct4) through binding to DR0 elements and harbors its intrinsic silencing function at the LBD (Cooney et al., 1998; Greschik et al., 1999; Lan et al., 2002; Yan et al., 1997). GCNF speciWcally binds to one of the two DR0 motifs in the Prm1 promoter and to the DR0 element in the Prm2 promoter (Fuhrmann et al., 2001). Antibody supershift assays show that endogenous GCNF isolated from mouse testes nuclear extracts and elutriated round spermatid nuclear extracts bind to both Prm1 and Prm2 promoters. A reporter gene bearing the protamine gene DR motif is potently repressed by GCNF. Thus, the Wndings demonstrate protamines as target genes silenced by GCNF. Silenced target gene transcription by GCNF for appropriate maintenance of physiologic functions is so far best reflected in its repression of the Oct4 gene transcription (Fuhrmann et al., 2001). The POU domain transcription factor Oct4 plays an essential role in the maintenance of embryonic stem cell potency and the establishment of the germ cell lineage. Oct4 is downregulated during gastrulation when embryonic stem cells diVerentiate, and its expression is subsequently conWned to the germ cell lineage. Consistent with this Wnding, expression of Oct4 is signiWcant in embryonic stem cells, carcinoma cells, and germ cells, whereas its expression is rapidly decreased in cells undergoing retinoid acid (RA)-induced diVerentiation. The requirement of GCNF for embryo viability and its dynamic expression proWles during embryogenesis imply that target genes critical for embryonic development (e.g. Oct4) may be subject to modulation by GCNF. In this regard, GCNF expression in both P19 embryonic carcinoma cells and mouse embryos inversely correlates with Oct4 expression. While Oct4 expression is shut oV during gastrulation, GCNF expression is signiWcantly up-regulated. Furthermore, dosage-dependent marked repression of Oct4 gene transcription by GCNF is observed in diVerentiating P19 embryonic carcinoma cells. The repression is mediated through a DR0 element in the Oct4 gene promoter. Mutation of each individual half-site to disrupt the DR0 element abolishes the GCNF-mediated silencing eVect, conWrming that speciWc homodimeric binding of GCNF to the Oct4 DR0 motif is a prerequisite for the repression. In addition, GCNF interacts with corepressor proteins NCoR and SMRT but not Alien in yeast and mammalian twohybrid systems and in vitro pull-down assays, indicating that repression of Oct4 gene by GCNF is mediated through recruitment of corepressors via

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direct protein–protein interaction. Studies in GCNF null mice have revealed that repression of Oct4 activity by GCNF is critical for conWning Oct4 expression to the germ line during pluripotent stem cell diVerentiation. In wild-type animals, Oct4 expression is restricted to primordial germ cells in the posterior of embryos at E8.5 to E8.75. In contrast, markedly altered expression of Oct4 is observed in the GCNF deWcient embryos, in which suppression of Oct4 expression in certain diVerentiating somatic cells is lost compared to the wild type. The Oct4 expression is no longer restricted to the germ cell lineage, while additional expression of Oct4 is detected in the putative hindbrain regions. Transrepression of target gene expression by GCNF has been observed in ERR
1-mediated gene activation (Yan and Jetten, 2000). Members of ERR subfamily and GCNF recognize similar response elements and display overlapped expression patterns. GCNF represses the ERR
1-induced gene transactivation through an ERR
1 response element. No protein–protein interaction is detected between GCNF and ERR
1, and repression is mediated at least in part through competitive binding of GCNF to the same ERR
1 response element. This also involves the participation of the corepressor, NCoR, which directly interacts with GCNF. Studies of the interaction between GCNF and NCoR have revealed some unique properties of GCNF. The hinge region and H3 and H12 of GNCF’s LBD are required for the interaction between GCNF and NCoR, for which the residues Ser246–Tyr247 in the hinge domain, Lys318 in H3, and Lys489–Thr490 in H12 are critical. Taken together, these Wndings support the important role of GCNF in embryonic development, neurogenesis, and reproduction through down-regulation of the expression of several target genes that are actively involved in these physiologic functions.

V. SHP: GENERIC HETERODIMERIC PARTNER-INHIBITING MULTIPLE NUCLEAR RECEPTOR PATHWAYS Short heterodimer partner (SHP) was initially cloned in an eVort to isolate interacting protein(s) for the mouse constitutive adrostane receptor (CAR) in yeast two-hybrid screening (Seol et al., 1996). As indicated by its name, SHP has been recognized as being able to interact and heterodimerize with a number of nuclear receptors, including MB67 (the human homologue of mouse CAR), thyroid hormone receptor (TR), retinoid acid receptor (RAR), retinoid X receptor (RXR), estrogen receptor (ER), glucocorticoid receptor (GR), and hepatocyte nuclear factor 4 (HNF4) (Borgius et al., 2002; Gobinet et al., 2001; Johansson et al., 1999; Klinge et al., 2001; Lee et al., 2000; Seol et al., 1996, 1998). The interaction of SHP with TR, RAR, or RXR is enhanced in the presence of their cognate ligand

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(Seol et al., 1996). The ability of SHP to down-regulate signaling pathways modulated by these multiple nuclear receptors indicates that the SHP-mediated repression represents a generic mechanism in the control of divergent physiologic functions. Several distinct mechanisms have been identiWed for SHP-induced gene silencing, including inhibition of DNA binding, competition with cofactor(s) for binding to nuclear receptors, and active repression through SHP’s intrinsic inhibitory activity. Furthermore, a critical physiologic requirement of SHP activity was demonstrated by studies using SHP-deWcient animal models. In addition, studies of the SHP gene promoter activity and its regulatory mechanism have provided further insights for the understanding of SHP function. This is of particular importance under the context of diVerential expression of a single protein because SHP may trigger complex changes in signal cascades due to its capacity to cross-talk with a wide range of nuclear receptors. SHP is an atypical orphan member in the nuclear receptor superfamily (Fig. 7). It contains a putative LBD but does not have a conventional DBD that is conserved among the nuclear receptors (Seol et al., 1996). No DNA binding activity has been recorded for SHP to date, and there is no ligand yet identiWed for its function. The SHP gene encodes a small nuclear protein that is composed of 273 amino acids in humans and 260 amino acids in mice. Studies of its genomic structure reveal that the SHP gene consists of two exons separated by a single intron, and the human SHP (hSHP) gene is located at chromosome 1p36.1 (Lee et al., 1998b). The hSHP gene contains a potential TATA box at its proximal 50 flanking region, and the

FIGURE 7. Function domains of SHP. SHP gene encodes a small protein containing 260 amino acids. The SHP protein contains a conserved LBD but lacks the conventional DBD domain. Three leucine-rich nuclear receptor boxes (NR boxes 1, 2, and 3) are located within its N-terminal, central, and C-terminal regions, where the amino acid sequences of each NR box with the LXXLL motif underlined are shown. SHP interacts with estrogen receptor (ER
or ER) in NR boxes 1 and 2, while its interaction with RXR, RAR, TR, HNF4, and GR depends on the NR box 2. SHP’s C-terminal LBD region harbors the domain for interaction with LXR and LRH-1 receptors (dotted line). The intrinsic repression domain of SHP is also located at the C-terminal (solid double lines).

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transcription start site is mapped to 32 nucleotides downstream of the TATA sequence. The expression of the SHP gene has been observed in various tissues, with high expression shown in the liver, heart, brain, adrenal gland, small intestine, and pancreas (Lee et al., 1998b). The wide expression pattern of the SHP gene, thus supports the physiologic relevance of this orphan receptor for interaction with numerous diVerent nuclear receptors under distinct biologic settings. Functionally, SHP inhibits binding of RAR:RXR heterodimer to its response element and represses retinoid acid-induced gene transactivation (Seol et al., 1996). The interaction of SHP with RAR or RXR may result in formation of novel heterodimers of SHP with RAR or RXR that are incapable of binding to the DNA. Similarly, SHP also exerts potent repression of the CAR-mediated or liganded TR-mediated gene activation by protein–protein interaction. Moreover, SHP interacts with RXR through its central domain and therein diVers from most other nonsteroid hormone receptors that use their C-terminal LBDs to dimerize with RXR (Seol et al., 1997). Furthermore, SHP, C-terminal domain harbors intrinsic repression activity, and this active inhibitory domain as well as its central RXRinteracting sequences are both required for SHP to achieve the maximal inhibition of the RXR-induced transactivation. The intrinsic C-terminal inhibitory region of SHP, however, does not interact with corepressor NCoR (Seol et al., 1997). This indicates that a distinct mechanism is employed in SHP-induced gene silencing since interaction and recruitment of NCoR is usually required for repression incurred by several nuclear receptors (i.e., unliganded TR or RAR/RXR and some orphan receptors) (McKenna et al., 1999). SHP participates in estrogen signaling pathways via its direct interaction with ER
and ER (Johansson et al., 1999; Seol et al., 1998). As observed for nonsteroid hormone receptors, the interaction of SHP with ERs is signiWcantly increased by estradiol (E2) but decreased by 4-OH tamoxifen. This suggests that agonist binding promotes formation of SHP-interacting surfaces in ERs. Consistent with this notion, SHP interacts with the ligand binding AF-2 domain of ER
to prevent its recruitment of the coactivator protein transcriptional intermediary factor 2 (TIF2) (Johansson et al., 1999). Similarly, SHP dose-dependently displaces the association of ER
/AF-2 domain with another coactivator: receptor-interacting protein 140 (RIP140) (Johansson et al., 1999). In agreement with the above studies, mutation of the ER
/AF-2 domain, or binding of an antagonist that is known to cause a distinct allosteric arrangement of AF-2, abolishes SHP binding to ER
. Consequently, SHP causes marked repression of E2-activated ER
transactivation and antagonizes TIF2-potentiated ER
-modulated target gene expression. SHP uses its N-terminal LXXLL-related (L represents leucine) motifs to bind to estrogen receptors, while its C-terminal LBD is not required for such interaction (Johansson et al., 2000). Similar leucine-rich

Gene Silencing

27

motifs, also called nuclear receptor (NR) boxes, are usually identiWed in the coactivator proteins as critical for their binding to the liganded AF-2 domains of nuclear receptors (McKenna et al., 1999). Cases in which SHP elicited marked inhibition of hepatocyte nuclear factor 4 (HNF4) and glucocorticoid receptor (GR)-induced gene activation, SHP also competed with coactivator proteins to bind HNF4 or ligand-bound GR (Borgius et al., 2002; Lee et al., 2000). Taken together, this evidence has revealed that competition with coactivators to occupy the same AF-2 domain through the leucine-rich motif is likely a generic mechanism in the SHP-mediated target gene inhibition for this class of receptors. The critical involvement of the SHP C-terminal repressive domain supports a two-step mode for SHP’s action, with initial displacement of a coactivator via its N-terminal LXXLL receptor-interaction motif. This is followed by a direct inhibitory eVect exerted by SHP’s C-terminal repression region (Lee et al., 2000). In addition, coexpression of SHP causes an intranuclear redistribution of GR molecules to cytoplasmic sites; however, such relocalization does not occur in the presence of inhibition-deWcient SHP mutants (Borgius et al., 2002). Thus, the tethering eVect of SHP may represent a novel mechanistic aspect in SHP-modulated gene repression. A direct interaction of SHP with peroxisome proliferator-activated receptors (PPARs) alpha and gamma and the subsequent regulation of PPAR-targeted genes, however, have shown several major diVerences. Whereas SHP binding to TR/RAR/RXR or ER/GR depends on their cognate ligand, its interaction with PPAR
or PPARg is not aVected by absence or presence of peroxisome proliferator ligands (i.e., Wy-14643) (Nishizawa et al., 2002). PPARg interacts with SHP through its DBD and the hinge region, rather than the ligand binding AF-2 domain; SHP strongly augments, rather than represses the PPARg-mediated gene activation at both basal and ligand-inducible conditions. The SHP-interacting domain within PPARg overlaps with its NCoR-interacting domain, and disassociation of NCoR binding from PPARg has been shown to contribute to the SHP-mediated PPARg activation. On the other hand, SHP up- or downregulates the genes involved in the peroxisomal -oxidation pathways through heterodimerization with PPAR
(Kassam et al., 2001). The dual regulatory mechanisms of SHP are likely attributed to the diVerent PPAR receptor subtypes and diVerent promoter contexts SHP recognizes. Critical physiologic contribution of SHP has been demonstrated in the coordinate regulation of bile acid synthesis pathways by SHP and several other nuclear receptors (Fig. 8). The catabolism of cholesterol into bile acids in the liver is essential for cholesterol homeostasis, and this process is tightly controlled through regulation of the activity of cholesterol 7
-hydroxylase (CYP7A1), the rate-limiting enzyme of this metabolic cascade (HoVmann, 1994). While the promoter activity of CYP7A1 is positively regulated by cholesterol derivative oxysterol, it is markedly repressed by bile acid.

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FIGURE 8. Control of bile acid synthesis by a regulatory cascade of nuclear receptors SHP, FXR, LRH-1, and LXR. Elevated cholesterol levels cause up-regulation of the CYP7A1 gene expression by binding of its oxysterol metabolites to RXR:LXR receptor heterodimmer in the CYP7A1 gene promoter. This is followed by increased catabolism of cholesterol and synthesis of bile acids as the end product. Accumulated bile acids in turn induce the transcription of the SHP gene by activation of the FXR receptor that binds as RXR:FXR heterodimer to an FXRE element in the SHP gene promoter. The SHP protein interacts with LRH-1 receptor and results in formation of an inactive SHP:LRH-1 heterodimer. This process represses the expression of the CYP7A1 gene and induces feedback control of the SHP gene due to the presence of LRH-1 binding sites in both gene promoters.

Mechanistically, the oxysterol receptor liver X receptor (LXR) heterodimerizes with RXR to activate the CYP7A1 gene transcription when binding to a cholesterol response element in the CYP7A1 gene promoter (Schoonjans and Auwerx, 2002; Stroup et al., 1997). This ligand-dependent LXR:RXR transactivation requires simultaneous binding of liver receptor homologue-1 (LRH-1) as an auxiliary competence factor to a LRH-1 element of the CYP7A1 promoter (Crestani et al., 1998; Gupta et al., 2002; Tu et al., 2000). In contrast to the direct feed-forward eVect of oxysterols through the promoter-bound RXR:LXR dimer (Peet et al., 1998), the feedback regulation of CYP7A1 activity by bile acid is indirect and is mediated by combined eVorts of SHP and farnesoid X receptor (FXR), LHR-1, and RXR (Goodwin et al., 2000; Lu et al., 2000; Makishima et al., 1999). Bile acid initially binds to its speciWc receptor FXR that dimerizes with RXR to recognize an FXR response element in the SHP gene promoter. The signiWcantly induced SHP gene expression by bile acid-bound RXR:FXR, subsequently causes marked repression of CYP7A1 gene promoter activity due to interaction of SHP with LRH-1. By forming a heterodimer with LRH-1, SHP is shown to inhibit both basal and oxysterol–RXR:LXR transactivated CYP7A1 gene transcription. Mice lacking the SHP gene displayed mild defects in bile acid homeostasis, whereas SHP/ or FXR/ null mice showed complete abrogation of SHP-mediated repression of CYP7A1 gene expression (Kerr et al., 2002; Wang et al., 2002). The down-regulation of CYP7A1 gene

Gene Silencing

29

expression by bile acid feeding is retained in the SHP null mice, indicating the existence of redundant or compensatory inhibitory pathway(s). Moreover, SHP exerts autoregulatory control of its own expression since its promoter contains a consensus LRH-1 binding site. This feedback regulation may provide a mechanism for attenuating the SHP-mediated gene silencing eVect. Therefore, the suppression of CYP7A1 gene transcription by SHP illustrates a complex regulatory network, which requires concerted participation of FXR, SHP, LHR-1, and RXR (Fig. 8). SHP has also been shown to negatively regulate human ATP-binding cassette transporter 1 (ABCA1) gene transcription through perturbation of LXR
:RXR-mediated gene transactivation in the presence of bile acid (Brendel et al., 2002). The activation of SHP expression by FXR:RXR, followed by its interaction with LXR
, accounts for the repression of human ABCA1 (hABCA1) gene expression by bile acid. The bile acidinduced repression of the sodium taurocholate cotransforting polypeptide (ntcp) gene, which is the principal hepatic bile acid transporter, is also attributed to SHP via its interaction with an RAR/RXR element of the ntcp promoter (Denson et al., 2001). Taken together, these Wndings demonstrate that formation of inactive heterodimers of SHP with other nuclear receptors may be a common mechanism in the control of genes involved in bile acid metabolism. The activation of SHP gene promoter activity by FXR and its autorepression through LRH-1 in the presence of elevated level of bile acid reveals that well-balanced SHP gene expression is important for accommodating a particular biologic function. Also, SHP gene transcription is activated by nuclear receptors SF-1 and fetoprotein transcription factor (FTF) (Lee et al., 1999c). At the molecular level, SF-1, FTF, and LHR-1 recognize the same Wve SF-1 binding elements identiWed in the SHP gene promoter because FTF and LHR-1 receptors are liver-speciWc close homologues of SF-1. Moreover, estrogen-related receptor gamma (ERRg), but not alpha (ERR
) or beta (ERR), was recently reported to strongly increase the SHP gene transcription by binding to one of the Wve SF-1 domains (Sanyal et al., 2002). This is consistent with the previous observation that the ERR isoforms
, , and g recognize both the estrogen response element and the SF-1 binding site. The lack of activation of the SHP gene by ERR
and ERR likely results from the association of these two receptors with putative corepressor protein(s). Although direct interaction of SHP with all three ERR subtypes has been demonstrated, SHP itself represses solely the ERRg-mediated activation of the SHP gene transcription. Therefore, cross-talk between SHP and ERRs has identiWed another novel autoregulatory loop in the control of SHP gene promoter activity. Because SHP is able to interact with a variety of nuclear receptors, a precise regulation of the SHP gene is particularly essential for its diVerential contribution in multiple divergent signaling pathways.

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VI. TR4 AND TR2: TESTICULAR RECEPTOR WITH HOMOLOGOUS BUT NOT REDUNDANT FUNCTIONS Testicular receptors 4 (also termed TAK1) and 2 (TR4 and TR2) are two evolutionarily conserved members within the nuclear receptor superfamily. Although they display structural features characteristic of the nuclear receptors, they are orphan receptors without a known ligand. The overlapping expression patterns of TR4 and TR2, as well as their functional participation in common signaling pathways, indicate that TR4 and TR2 may participate in modulation of the same sets of target genes. Both receptors exert dual regulatory mechanisms and achieve a positive or a negative eVect depending on the speciWc target genes they recognize. In TR4/TR2-mediated gene silencing events, active repression and transrepression of multiple hormone-regulated pathways are observed. Several mechanisms are operative in the inhibitory process, including competition with nuclear hormone receptors for the same cis-elements, impairment of hormone receptor binding activities, competition for coactivator proteins, and modiWcation of chromatin structures. In addition, diVerences have also been noted between TR4 and TR2 despite their striking similarities, which indicates that these two orphan receptors may not be physiologically redundant. The initial cloning of the human TR4 gene from the prostate and testes and the TR2 gene from the prostate was followed by identiWcation of their homologues in diVerent species, including rats, mice and Drosophila (Chang and Kokontis, 1988; Chang et al., 1994; Hirose et al., 1994; Lee et al., 2002; Sanyal et al., 2003). In humans and rodents, several variants of TR4 and TR2 gene transcripts are generated by alternative splicing (Chang and Kokontis, 1988; Chang et al., 1989; Yoshikawa et al., 1996a,b). Human full-length TR4 and TR2 are composed of 615 and 603 amino acids, respectively, and display high homology at their amino acid sequences, with 82% and 65% identities at their respective DBDs and LBDs (Fig. 9). At the genomic level, the human TR4 and TR2 genes are located at chromosome 3p24 and 12q22, respectively (Lee et al., 1995; Lin et al., 1998). These receptors are expressed in multiple tissues of adult mammals, and their abundance varies signiWcantly (Chang and Kokontis 1988; Chang et al., 1989; Hirose et al., 1994). Expression is highest in the prostate and testes, and high levels of expression are present in the adrenal gland, spleen, and thyroid gland. During embryonic development, TR4 and TR2 are predominantly expressed in the central nervous system during proliferation and diVerentiation in embryonic neurogenesis (Lee et al., 1996; Young et al., 1998). In addition, the expression of TR4 and TR2 is influenced by a number of factors (i.e., retinoid, p53 and others), which may contribute to a dynamic regulatory network of their function (Inui et al., 1999; Lee and Wei, 1999; Lin and Chang, 1996).

Gene Silencing

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FIGURE 9. TR2 and TR4: Functional domains and repressive mechanisms. Human TR2 and TR4 are highly homologous. Active repression of target gene expression by TR2 and TR4 is mediated by the DBD and LBD (dotted lines). The DBD also serves as an interface for TR2 to associate with classes I and II histone deacetylases (HDACs 3 and 4). Heterodimerization between TR2:TR4 is dependent on the region of helices 10 to 12 (H10–12) of TR2. TR2 or TR4 represses the hormone-induced gene activation by competing for binding with RAR, RXR, TR, or VDR receptors, decreasing the binding activity of ER through protein–protein interaction, or titrating out of coactivator RIP140 from PPAR
. In addition, TRA16 functions as corepressor for TR4-induced gene transactivation.

The binding properties of TR4 and TR2 have been intensively investigated since the original cloning of these two receptor genes. TR4 binds eVectively to synthetic or natural DNA response elements that are composed of direct repeats (DR) of core motif PuGGTCA separated by zero to Wve nucleotides (DR0–5) (Hirose et al., 1995a). In contrast, TR4 binds poorly to palindromic or inverted repeat sequences. Similar binding kinetics are observed for TR2; it prefers to bind to direct-repeat motifs with various lengths of spacers. Binding aYnities follow the order of DR1>DR2>DR4¼DR5¼DR6>DR3 (Lin et al., 1995). TR4 and TR2 usually bind to a DR element as homodimers. Active repression by TR4 or TR2 through various DR elements has been shown in regulation of many viral and cellular genes. Both TR4 and TR2 exert marked repression of early and late SV40 promoters via binding to a DR2-type response element, and TR2 suppresses the promoter activities of human erythropoietin (EPO) and hepatitis B virus genes (Lee and Chang, 1995; Lee et al., 1996; Yu and Mertz, 1997). Heterodimeric binding of TR4 and TR2 to a DR5-containing reporter gene elicits much stronger repression when compared to the action by either homodimer alone (Lee et al., 1998a). In this case, protein–protein interaction between TR4 and TR2 involves helix 10 (H10) of TR2. In addition, a 540-kDa protein complex containing TR4:TR2 heterodimer as its core component represses embryonic and fetal globin gene

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transcription in deWnitive erythroid cells (Tanabe et al., 2002). Based on the coexistence of TR4 and TR2 at certain physiologic stages, heterodimerization of these two receptors may provide a more eVective inhibitory mechanism in the control of speciWc target gene expression. Although initial studies did not detect binding of TR4 or TR2 to a single half-site of the core element, speciWc monomeric binding of TR4 to an AGGTCA element results in suppression of human steroid 21-hydroxylase gene expression (Lee et al., 2001). The notion that TR4 and TR2 play signiWcant roles in down-regulation of hormonal signaling pathways is derived from evidence that the various DR elements recognized by TR4 and TR2 also serve as consensus binding sites for nonsteroid nuclear hormone receptors. TR4 and TR2 signiWcantly repress RA-transactivated cellular retinal-binding protein II (CRBPII) and RAR gene transcription via potent binding of TR4 or TR2 to the DR1 or DR5 motif (Chinpaisal et al., 1997; Lin et al., 1995). TR4 and TR2 do not interact directly with RXR or RAR, and the repression is attributed to competitive occupancy of the same response elements by TR4 or TR2 homodimer or putative TR4:TR2 heterodimer and RAR
:RXR
heterodimer. TR2 represses T3-activated reporter gene activity by competitive binding with TR
:RXR
heterodimer to a DR4 element (Hirose et al., 1995a). This is also the case for the TR4-mediated repression of vitamin D3induced 25-hydroxyvitamin D3 24-hydroxylase gene activity at a DR3 motif (Lee et al., 1999b). In addition, silencing of PPAR
-modulated transactivation of rat enoyl-CoA hydratase and peroxisomal fatty acyl-CoA oxidase gene expression by TR4 is caused by competition of TR4 with PPAR
:RAR
heterodimer (Yan et al., 1998). A mechanism involving titration out of RXR from PPAR
:RXR
is excluded since TR4 does not interact with PPAR
or RXR
. Furthermore, the PPAR ligand 8(S)-hydroxy-eicosate-traenoic acid strongly promotes the recruitment of coactivator protein RIP140 to PPAR
, while it decreases PPAR
association with corepressor SMRT. TR4 can directly interact with RIP140, but not with SMRT, and competes with PPAR
for binding to RIP140 (Yan et al., 1998). Thus, at least two cooperative mechanisms participate in TR4-induced down-regulation of PPAR responsive genes: competition with PPAR
:RXR
heterodimer for binding to the PPAR response element and competition for binding to coactivators, such as RIP140. In addition, TR4 and TR2 display diVerential regulatory function in the PPAR
signaling pathways. In human HaCaT keratinocytes, TR4, but not TR2, causes marked inhibition of the PPAR
-activated reporter gene activity in the presence of Wy-14643, a peroxisome proliferator ligand (Inui et al., 2003). In addition, the endogenous expression of TR4 protein is induced by Wy-14643, whereas TR2 protein expression is decreased under the same condition. Hence, it can be proposed that selective up-regulation of

Gene Silencing

33

TR4 but not TR2 expression, followed by subsequent increase of TR4’s repressive activity, may be required for Wne-tuning of target genes in response to the agonist. Furthermore, TR2 is found to directly interact with histone deacetylases 3 and 4 (HDAC3 and HDAC4) (Franco et al., 2001). Functionally, the TR2-repressed RAR gene activation by RA is signiWcantly compromised by trichostatin A (TSA), a speciWc histone deacetylase inhibitor, which suggests that HDACs may work cooperatively with TR2 in the inhibition of gene expression. Repression of target gene expression by TR4 and TR2 via mechanism(s) distinct from direct competitive DNA binding has also emerged. Although TR2 or TR4 does not bind palindromic repeat motifs that include the binding sites for estrogen receptors (ER
and ER), it shows that TR4 and TR2 repress the ER-transactivated gene expression by direct protein–protein interaction of TR4 or TR2 with ER (Shyr et al., 2002a,b). Such interaction prevents the formation of an active ER homodimer and abrogates ER binding to the estrogen response element. Functionally, TR4 markedly represses two estrogen responsive genes, cyclin D1 and pS2, in human mammary gland carcinoma cells (MCF-7), and the ER-mediated cell growth and proliferation is suppressed in cells harboring stable transfected TR4. Similar Wndings have been also observed for TR2. Also, cross-talk of TR4 with other nuclear receptors has been noted. Interaction of TR4 with androgen receptor (AR) is found to trigger bidirectional mutual repression on TR4- and AR-modulated target gene expression (Lee et al., 1999a). While AR represses TR4-activated expression of ciliary neurotrophic factor (CNTF) receptor gene, TR4 exhibits potent repression of AR-activated promoter activities of mammary tumor virus (MMTV) and prostate-speciWc antigen (PSA) genes in the presence of AR ligand dihydrotestosterone (DHT). The repression by TR4 is shown to be AR-speciWc since it does not aVect the glucocorticoid receptor- or progesterone receptor-induced transactivation of the MMTV gene. Convergence of TR4- and AR-regulated pathways may have signiWcant physiologic relevance because TR4 is most abundantly expressed in the prostate, the major target site of androgen action. TR4 interacts with hepatocyte nuclear receptor 4 (HNF4) to repress HNF4-transactivated hepatitis B virus (HBV) core promoter activity through an interaction that is mediated by a DNA-bound form of TR4 at a DR1 motif (Lin et al., 2003). Since TR4 does not aVect the HNF4 binding activity to the HBV promoter, a diVerent mechanism other than cross-talk between TR4 and ER/AR is operative in this case. In addition to the potent repression of multiple target gene expression by TR4 and TR2, these two orphan receptors also exert positive transcriptional control of several genes. TR4 and TR2 activate reporter gene activity through a synthetic DR4 thyroid hormone response element (DR4-TR) (Chang and Pan 1998; Lee et al., 1997). By binding competitively to this motif, the TR2-induced activation antagonizes the inhibitory eVect of the

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reporter gene activity by the unliganded TR
receptor. In the presence of T3, while an activating form of ligand-bound TR
is induced, TR2 exerts an additive eVect on T3-activated gene expression. Moreover, TR4 up-regulates the
-myosin heavy chain and rat S14 genes through a DR4-like element, the human luteinizing hormone receptor gene through an imperfect DR0 element, as well as the human CNTF (ciliary neutrotrophic factor) receptor gene expression via a DR1 domain (Lee et al., 1997; Young et al., 1997; Zhang and Dufau, 2000). CNTF elicits a feed-forward regulatory eVect by increasing the expression of both TR4 and TR2 proteins, resulting in enhanced TR4/TR2 activation of the CNTF receptor gene. The two orphan receptors are coexpressed with the CNTF receptor in many developing neural structures, indicating their active participation in neurogenesis. Although the mechanism of TR4/TR2-induced gene activation is poorly understood at this stage, current evidence suggests that these orphan receptors may adapt distinct conformations depending on the speciWc promoter context. Gel mobility shift assays of TR4 to a DR3/VDR element as a repressor and to a DR4/TR element as an activator reveal the formation of diVerent antibody supershift complexes of TR4 when it binds to these two diVerent motifs (Lee et al., 1999b). Moreover, proteolytic analyses of the DNA-bound TR4 protein have revealed distinct peptide digestion patterns, indicating the diVerent conformations of TR4 receptor on these elements. It is possible that diVerent allosteric structures of TR4 may favor its selective interaction with corepressors or coactivators, thereby eliciting opposite eVects through a DR3/VDR or a DR4/TR element. Furthermore, a novel 16-kD TR4-interacting protein, TRA16, has been recently identiWed through yeast two-hybrid screening and has been shown to act as a repressor of TR4-mediated gene activation (Yang et al., 2003). Mechanistically, TRA16 decreases the binding activity of TR4, blocks formation of TR4 homodimer, and consequently abrogates TR4-induced gene activation. In summary, the orphan nuclear receptors TR4 and TR2, through recognition of various DR motifs located in a number of viral and cellular genes, play an important role in the control of multiple biologic functions.

VII. CONCLUSIONS IdentiWcation of the orphan receptors COUP-TFs, DAX-1, GCNF, SHP, and TR4 and 2, as well as the multiple processes by which they negatively regulate their target genes, has revealed the molecular mechanisms by which these receptors operate in the inhibitory control of cell functions. These Wndings and studies in knockout animals (COUP-TFs, DAX-1, GCNF, and SHP) and in patients with naturally occurring mutations (DAX-1) have provided direct evidence linking the orphan receptors’ inhibitory actions to physiologic states and pathologic conditions.

35

Gene Silencing

Despite the large amount of knowledge that has been accumulated in this Weld of research, much remains to be elucidated. The manner in which multiple mechanisms are coordinated in a tissue- or cell-speciWc manner to achieve concise regulatory control has yet to be explained. Further studies by tissue- and cell-speciWc knockouts of certain orphan receptors to avoid embryonic lethality will clarify additional speciWc functions and provide insights into novel regulatory pathways. The possible involvement of speciWc unidentiWed corepressor or coadaptor proteins still needs to be determined, and the relevance of orphan receptor-mediated repression with epigenetic control of gene expression through histone modiWcation and remodeling and DNA methylation events requires detailed analysis. Because most evidence is based on studies of TATA-box genes, which diVer in their regulation from TATA-less genes, further study of the silencing mechanisms operative for the latter could provide new insights into their functional control by orphan receptors. Moreover, identiWcation of the putative ligands for these orphan receptors could lead to the discovery of novel therapeutic pathways and targets for drug development. Finally, characterization of the regulatory cascades governing their own expression will delineate the complex networks of cross-talk between COUP-TFs, DAX-1, GCNF, SHP, or TR2 and 4 and other transcription factors involved in repression of target gene expression.

REFERENCES Achatz, G., Holzl, B., Speckmayer, R., Hauser, C., Sandhofer, F., and Paulweber, B. (1997). Functional domains of the human orphan receptor ARP-1/COUP-TFII involved in active repression and transrepression. Mol. Cell. Biol. 17, 4914–4932. Achermann, J. C., Ito, M., Silverman, B. L., Habiby, R. L., Pang, S., Rosler, A., and Jameson, J. L. (2001a). Missense mutations cluster within the carboxyl-terminal region of DAX-1 and impair transcriptional repression. J. Clin. Endocrinol. Metab. 86, 3171–3175. Achermann, J. C., Meeks, J. J., and Jameson, J. L. (2001b). Phenotypic spectrum of mutations in DAX-1 and SF-1. Mol. Cell. Endocrinol. 185, 17–25. Agoulnik, I. Y., Cho, Y., Niederberger, C., Kieback, D. G., and Cooney, A. J. (1998). Cloning, expression analysis, and chromosomal localization of the human nuclear receptor gene GCNF. FEBS Lett. 424, 73–78. Alland, L., Muhle, R., Hou, H., Potes, J., Chin, L., Schreiber-Agus, N., and DePinpo, R. A. (1997). Role for NCoR and histone deacetylase in Sin3-mediated transcriptional repression. Nature 387, 49–55. Altincicek, B., Tenbaum, S. P., Dressel, U., Thormeyer, D., Renkawiz, R., and Baniahma (2000). Interaction of the corepressor Alien with DAX-1 is abrogated by mutations of DAX-1 involved in adrenal hypoplasia congenita. J. Biol. Chem. 27, 7662–7667. Arango, N. A., Lovell-Badge, R., and Behringer, R. R. (1999). Targeted mutagenesis of the endogenous mouse Mis gene promoter: In vivo deWnition of genetic pathways of vertebrate sexual development. Cell 99, 409–419. Avram, D., Ishmael, J. E., Nevrivy, D. J., Peterson, V. J., Lee, S. H., Dowell, P., and Leid, M. (1999). Heterodimeric interactions between chicken ovalbumin upstream promotertranscription factor family members ARP-1 and EAR2. J. Biol. Chem. 274, 14331–14336.

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